This invention relates to an improved method of qualitative and quantitative analysis, and more particularly to an improved method for resolving overlapped chromatographic peaks using multichannel detection techniques.
A recurring challenge facing the qualitative analysist is "separation," or accurately separating and identifying the individual components that are present in a mixture or substance having an unknown composition. Chromatography is one example of a well-known separation method that can be used to separate and analyze mixtures of chemical substances. Examples of other forms of separation methods include distillation, foam fractionation, adsorption, sublimination, molecular sieves, ion exchangers, membrane filtration, electro-dialysis, thermal diffusion and mass spectrometry.
Simply stated, chromatrography is based upon the time it takes a specific component or substance to elute or migrate through a porous medium. For example, suppose a sample substance containing a mixture of unknown, distinct components A, B, & C is introduced into one end of a column or channel that is filled with a porous medium (the porous medium often being referred to as the stationery phase). The physical and chemical properties of each of the components A, B, and C, in conjunction with the physical and chemical properties of the porous medium, cause a specific migration time (often referred to as the "retention time") to be associated with each component as it flows to the other end of the column or channel. Thus, whereas all three components are placed or injected into one end of the column (or channel) simultaneously, component A may arrive at the other end of the column one minute after injection, component B three minutes after injection, and component C five minutes after injection. By knowing in advance which components migrate through the column within the one minute time window, the three minute time window, and the five minute time window (which knowledge is generally obtained experimentally using known compounds and, once obtained for a given column and porous medium, is cataloged for future reference), the unknown compounds A, B, and C can be identified. This identification assumes, of course, that a suitable detector is employed at one end of the column to detect the arrival time of each compound, and that migration time data has been previously obtained for each compound present. It also assumes that two distinct compounds do not share the same migration time, i.e., arrive at the detector within the same time window. The detector can also be used to quantitatively measure the relative concentrations of each compound within the mixture.
Chromatography may theoretically be used to analyse gases, liquids, and solids. A widely used form of chromatography in practice is gas-liquid chromatography, wherein the stationary phase is a liquid, and the mobile phase--that phase that either carries or contains the mixture of unknown compounds--is a gas. Because of its popularity, gas-liquid chromatography is often referred to as simply gas chromatography, or "GC". However, other forms of chromatography, such as liquid-liquid chromatography, are not uncommon.
While gas chromatography is an excellent tool for the separation, detection, and quantification of the components of a complex mixture, it is not, by itself, a good tool for quantitative identification. This is because there may be many thousands of compounds that may share the same retention time, thereby eluting or migrating within the same time period. The use of two or more columns, each having different retention indexes, has proven to be a viable solution to this common retention-time problem only for simple mixtures or pure compounds. Thus, because a large number of compounds share the same retention-time, effective qualitative analysis requires either a wealth of a prior information concerning what compounds are most likely to be present (thereby allowing the exclusion of compounds not likely to be present) or the combination of gas chromatography with some other separation method. In the latter case, confirmation by two or more methods is desirable. A common choice of an additional separation technique to supplement chromatography is mass spectrometry, or "MS". This is because of its high sensitivity and relatively specific spectral information.
In its simplest form, mass spectrometry consists of taking the compound to be identified and, in the gas phase, breaking it up into its constituent ions. These ions are then sorted and counted as a function of their mass. The output of a mass spectrometor thus provides specific data concerning the relative concentrations of mass-identified ions within the compound. As such, it serves as a unique "finger-print" of the compound, and can effectively be used to identify the presence of the compound within the sample mixture being analysed.
Combining mass spectrometry with gas chromatrography--a combination known as GC-MS--provides a powerful analytical tool. The gas chromatography provides an initial separation of the compounds as a function of retention time. The mass spectrometer then provides a positive identification of one of several compounds that could exhibit the measured retention time. In practice the GC-MS method is realized by connecting the output of the gas chromatographic column, through a suitable interface, to the input of a mass spectrometer. The compound that elutes or migrates from the chromatographic column within a given time window, which compound could be one of thousands of possible compounds, is then immediately exposed to the mass spectrometer for further analysis. Based on the mass spectrum obtained through this analysis, the compound (through a comparison of its measured spectrum to a catalog of spectrums) can usually be positively identified.
Despite the powerful analytical tool provided by the GC-MS combination (and similar combinations, such as gas or liquid chromatography coupled with infrared spectrum analysis, referred to as GC-IR or LC-IR respectively), a serious deficiency is encountered when two or more compounds having the same, or very close to the same, retention times are simultaneously present in the mixture. This condition is termed "overlapped chromatographic peaks" and the problem of identifying the compounds that are present within the overlap region is the specific problem addressed by this invention.
The problem of resolving overlapped chromatographic peaks can be more fully appreciated, especially by those unskilled in the art, through reference to a simple "bomb-blast" analogy. If a desk, for example, is blown up into a "million" pieces by a bomb blast, and if most of the pieces of the desk can be located and collected, a careful analysis of the pieces will generally reveal the identity of the original item, i.e., a desk. If, on the other hand, a desk, table, and chair are all simultaneously blown up by a bomb blast, and even if most of the resulting pieces are located and collected, it would be an almost impossible task to determine the identity of the original items, especially if there were no prior knowledge as to the number of items originally present. Moreover, even if it were known that there were originally three items, one of which was a desk, it would be extremely difficult to determine which pieces belonged to the desk and which did not, especially if the desk, table and chair were all made from the same or similar material.
The above bomb-blast analogy reflects, in a very simplified way, the problem facing an analyst who is attempting to identify the compounds present in an overlapped chromatographic peak, using, for example, mass spectrometry. That is, two or more compounds are present within the peak. When these compounds are "blasted apart" by breaking them up into their constituent ions, the analyst has no accurate method of determining which ions belong to which compound, unless he has prior information concerning which compounds are present. Thus, no single identifying spectrum can be obtained.
Prior art methods for interpreting spectral data obtained from overlapped chromatographic peaks have been fraught with limitations. These methods, especially as used in connection with GC-MS, have in common the recognition that the spectral pattern of a particular compound from a multi-channel detector will rise and fall in unison when one compound elutes. Accordingly, if the chromatographic data indicates the presence of two or more peaks within a given time window, one prior art separation method presumes that one peak belongs to only one compound, that one being the "sharpest" within the particular time window. After identifying the sharpest peak, the method then identifies the spectral channels which follow the same time behavior using a correlation criterion. All the spectral channels which follow reasonably well are assigned to that single component having the sharpest peak. Any channels which do not correlate are presumed to below to a different component. By subtracting out the spectral data following the time behavior of the sharpest peak, it thus becomes possible to unscramble the mixed spectral data.
The above described prior art method may be referred to as the "template approach" because it assumes that the shape of the over-lapped peak is known. This shape--or the "template"--is then presumed to belong to the same component and subtracted out. The main problem associated with this approach is in defining the "template." That is, it is difficult to accurately measure the "sharpest peak," especially if a truly complex mixture is being analysed (one in which two or more compounds are likely to be truly overlapped, having little separation between them). Also, it is highly unlikely that each compound in a particular time window can be represented by totally resolved spectral channels.
An alternative method for resolving overlapped data peaks consists of comparing the spectra of mixtures resulting from the overlapped peaks to a library of spectra of compounds that could be present. A probability based matching system using a large collection of reference spectra then determines which compound is most likely present in the mixture, subtracts the spectrum of that compound from the mixture data, and the remaining spectra are matched to the library to see whether any other compounds can be identified. This process continues until no new compounds can be matched, or until the remaining data drops below the noise level. The problems associated with this approach relate to having a priori information concerning what compounds are likely to be present. Moreover, there are always inefficiencies related to the handling of large amounts of data. Even with modern computer based systems, the data must be gathered, cataloged, and entered into the system, and once there, it must be retrievably stored. All this requires a considerable investment of time and money. Further, it is unlikely that all the compounds in a sample will be present in the library. In addition, what data is present in the library will be biased with the "signature" of the instrument through which the data was obtained.